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A number of different positive and negative controls must be included to support the validity of the staining pattern and to exclude experimental artefacts. Furthermore, detailed record keeping is key to ensuring consistent performance as variation in experimental conditions and the condition of the tissue itself may impact the reproducibility of staining.
A section from a tissue known to express the protein of interest. A positive result from the positive control, even if the samples are negative, will indicate that the procedure is working and optimized. It will verify that any negative results are valid.
A section from a tissue known not to express the target antigen. This is to check for non-specific signal and false positive results.
Endogenous tissue background control
A section from the tissue before applying the primary antibody. Certain tissues have inherent properties that result in background staining, which could affect the interpretation of results. For example, certain tissues contain endogenous fluorescent molecules that could be confused for positive staining during fluorescent IHC. The tissue should be checked under the microscope using either fluorescent or bright-field illumination (for fluorescent or chromogenic labels, respectively) to ensure that there is no endogenous background.
No primary antibody control
Tissue is incubated with the antibody diluent alone and no primary antibody, followed by incubation with secondary antibodies and detection reagents. This ensures that staining is produced from detection of the antigen by the primary antibody and not by the detection system or the specimen.
Tissue is incubated with the antibody diluent and a non-immune antibody of the same isotype and at the same concentration as the primary antibody, followed by incubation with secondary antibodies and detection reagents. This control checks that the observed staining is not caused by non-specific interactions of the antibody with the tissue. Any background staining observed with this control should be negligible and distinct from specific staining. This control can be used when working with monoclonal primary antibodies.
Tissue is incubated with pre-absorbed antibody instead of the primary antibody, followed by incubation with secondary antibodies and detection reagents. As this control is performed to demonstrate that the antibody binds specifically to the antigen of interest, little or no staining is expected. Pre-absorbed antibody may be produced by overnight incubation of the antibody at 4°C with a large molar excess (10-fold) of the immunogen. Absorption controls are more reliable if the immunogen is a peptide, as antibody-protein immunogen mixtures may themselves cause high background staining in tissues due to non-specific interactions between the protein and the tissue.